The studies described in this proposal will aid in describing the pathway of protein degradation in heart muscle, in evaluating the physiological role of hormonal and non-hormonal factors that regulate proteolysis and in determining the biochemical mechanisms by which these regulatory factors act. An important facet of these studies will involve localization of protease to heart muscle cell as compared to non-muscle cells in the tissue. The objectives are 1) to evaluate the physiological role of leucine, insulin, epinephrine and glucagon in regulation of protein degradation in heart muscle in the working heart preparation that is perfused under conditions simulating levels of ventricular pressure development and substrate and hormone availability that are found in vivo, 2) to purify the proteases of heart muscle cells that have neutral-alkaline pH optima to homogeneity and to characterize them by studies of substrate specificity and active-site inhibitors, 3) to study uptake of native and denatured proteins and proteolytic products resulting from the action of neutral-alkaline proteases by heart muscle cell lysosomes, and 4) to investigate the energy requirement for protein degradation. The hypothsis that will be tested is that protein degradation in heart muscle cells involves hydrolysis of peptide bonds in a single proteolytic pathway that includes proteases that are active at neutral or mildly alkaline pH and are located in the cytoplasm followed by engulfment and digestion within lysosomes.